这项工作是由Luminex公司。

<ol>
	<li>Fulton, J. R., McDade, R. L., Smith, P. L., Kienker, L. J., Kettman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+multiplexed+analysis+with+the+FlowMetrix+system.">Advanced multiplexed analysis with the FlowMetrix system.</a> Clinical Chemistry. 43, 1749-1756 (1997).</li><li>Carson, R. T., Vignali, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantation+of+15+cytokines+using+a+multiplexed+flow+cytometric+assay.">Simultaneous quantation of 15 cytokines using a multiplexed flow cytometric assay.</a> J. Immunol. Methods. 227, 41-52 (1999).</li><li>Peck, D., Crawford, E. D., Ross, K. N., Stegmaier, K., Golub, T. R., Lamb, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+gene+expression+signature+analysis.">A method for high-throughput gene expression signature analysis.</a> Genome Biol. 7, 61 (2006).</li><li>VanDerMeid, K. R., Su, S. P., Krenzer, K. L., Ward, K. W., Zhang, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+extract+cytokines+and+matrix+metalloproteinases+from+Schirmer+strips+and+analyze+using+Luminex.">A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.</a> Mol Vis. 17, 1056-1063 (2011).</li><li>Liu, J., Kibiki, G., Maro, V., Maro, A., Kumburu, H., Swai, N., Taniuchi, M., Gratz, J., Toney, D., Kang, G., Houpt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+reverse+transcription+PCR+Luminex+assay+for+detection+and+quantitation+of+viral+agents+of+gastroenteritis.">Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis.</a> J. Clin. Virol. 50, 308-313 (2011).</li><li>de Jager, W., Prakken, B. J., Bijlsma, J., WJ Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+multiplex+immunoassay+performance+in+human+plasma+and+synovial+fluid+following+removal+of+interfering+heterophilic+antibodies.">Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.</a> J. Immunol. Methods. 300, 124-135 (2005).</li><li>Codorean, E., Nichita, C., Albulescu, L., Raducan, E., Popescu, I. D., Lonita, A. C., Albulescu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+xMAP+and+ELISA+cytokine+profiles;+development+and+validation+for+immunotoxicological+studies+in+vitro.">Correlation of xMAP and ELISA cytokine profiles; development and validation for immunotoxicological studies in vitro.</a> Roum. Arch. Microbiol. Immunol. 69, 3-19 (2010).</li><li>de Jager, W., te Velthuis, H., Prakken, B. J., Kuis, W., Rijkers, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+15+human+cytokines+in+a+single+sample+of+stimulated+peripheral+blood+mononuclear+cells.">Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.</a> Clin. Diagn. Lab. Immunol. 10, 133-139 (2003).</li><li>DuPont, N. C., Wang, K. H., Wadhwa, P. D., Culhane, J. F., Nelson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+comparison+of+Luminex+multiplex+cytokine+analysis+kits+with+ELISA:+Determinations+of+a+panel+of+nine+cytokines+in+clinical+sample+culture+supernatants.">Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants.</a> J. Reprod. Immunol. 66, 175-191 (2005).</li><li>Richens, J. L., Urbanowicz, R. A., Metcalf, R., Corne, J., O'Shea, P., Fairclough, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+validation+and+comparison+of+multiplex+cytokine+kits.">Quantitative validation and comparison of multiplex cytokine kits.</a> Journal of Biomolecular Screening. 15, 562-568 (2010).</li><li>Rizzi, G., Zhang, Y. J., Latek, R., Weiner, R., Rhyne, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+development+of+a+Luminex-based+assay+for+the+detection+of+human+IL-23.">Characterization and development of a Luminex-based assay for the detection of human IL-23.</a> Bioanalysis. 2, 1561-1572 (2010).</li></ol>

这项工作是在Luminex公司与Luminex公司制造的设备。 研发系统与EMD的微孔Luminex公司的战略合作伙伴;许可的开发和商品化基于多重XMAP的检测。

酶联免疫吸附试验转换的Luminex XMAP平台，可以简单，只要代链霉亲和素辣根过氧化物酶（SA-HRP）的，在一个典型的链霉，藻红蛋白（SA-PE）的ELISA试剂盒和性能优化。对于那些谁想要建立XMAP免疫，从地上爬起来，这可以用一个简单的协议，也使快速，多重抗体对评价完成。为XMAP检测试剂很容易被准备使用XMAP抗体偶联试剂盒夫妇指定的捕获抗体MagPlex低浓度微。使用低浓度微减少，而高浓度微提供相同的检测性能检测方法开发的成本。的准备再加MagPlex微所需的时间量是大约3个小时，这是速度远远超过了22 - 24小时大衣以及酶标板治疗涂井。的XMAP法的性能也优于酶联免疫吸附检测（&lt;4皮克/毫升与31皮克/毫升）和动态范围（&lt;4皮克/毫升至8000皮克/毫升比16的限制条款皮克/毫升至1000皮克/毫升）。板读者有一个有限的外径范围是3或4外径，限制检测的动态范围为上限。 毫无疑问，并非所有的抗体，将工作在ELISA格式，并非所有的抗体，在酶联免疫吸附试验工作，随时转让的XMAP检测格式。然而，因为XMAP检测可复用（例如，同时运行），它是可能的，以评估数的捕获和检测抗体组合，同时使用一个试验以确定最佳的一双。这个过程中可以节省大量的时间和试剂相比，酶联免疫吸附试验发展过程中，时间是有限的一对评价。如果两个或两个以上的抗体对执行等价;其他参数的检测被认为是对的（例如，可用性，成本等），以确定适宜。 除了与XMAP检测以提高​​检测性能和灵活性，也有显着的成本节约。建议的抗体数量所需的外套单井酶标板是400ng，而用于一个XMAP检测珠所需的数量大约是7.5纳克。因此，所需金额为酶联免疫吸附试验的抗体，将提供超过50个测试结果，如果用在XMAP法。对于涉及珍贵样品的应用，XMAP也有一个显着的优势。建议的酶联免疫吸附试验的样本量为100μL，而为XMAP检测所需的数量可以是一半，或更少。 总之，酶联免疫吸附试验的Luminex XMAP平台转换是简单，高效，节约成本，同时生产具有优异的动态范围和灵敏度的检测。

<table border="1"><tbody><tr><td> 描述 </td><td> 供应商 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>人类TNF-α/TNFSF1ADuoSet ELISA试剂盒</td><td> R＆D系统</td><td> DY210 </td><td>包括单克隆抗体和生物素耦合多克隆抗体和重组肿瘤坏死因子-α蛋白标准</td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> abcam </td><td> ab18696 </td><td>捕获抗体，克隆CH8820 </td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> abcam </td><td> ab16166 </td><td>生物素耦合检测抗体，克隆AS1的</td></tr><tr><td>肿瘤坏死因子α蛋白</td><td> abcam </td><td> ab9642 </td><td>重组肿瘤坏死因子-α蛋白标准</td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> ñovus </td><td> NBP1-50115 </td><td>捕获抗体，克隆4H31 </td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> Novus公司</td><td> NB100-78162 </td><td>生物素耦合检测抗体，克隆MAb11 </td></tr><tr><td>肿瘤坏死因子α蛋白</td><td> Novus公司</td><td> NBC1-18460 </td><td>重组肿瘤坏死因子-α蛋白标准</td></tr><tr><td>肿瘤坏死因子-α单克隆抗体</td><td> EMD的微孔</td><td> MAB1141 </td><td>捕获抗体，克隆3C7.2 </td></tr><tr><td>肿瘤坏死因子-α多克隆抗体</td><td> EMD的微孔</td><td> 654250 </td><td>兔多克隆抗体检测抗体</td></tr><tr><td>链霉亲和素 - 藻红蛋白</td><td>苔</td><td> SAPE-001 </td><td>荧光记者点阵仪XMAP含量的试剂</td></tr><tr><td> MAGPIX W / xPONENT软件</td><td> Luminex公司</td><td>马GPIX-XPONENT </td><td> Luminex液相仪器</td></tr><tr><td> XMAP抗体偶联（ABC）套件</td><td> Luminex公司</td><td> 40-50016 </td><td>包括EDC试剂，磺酸基-NHS的试剂，激活缓冲，洗涤缓冲液，1.5 ml反应管，一次性移液器</td></tr><tr><td> MagPlex微球，低浓度</td><td> Luminex公司</td><td> MC10012-ID，MC10013-ID，MC10014-ID，MC10015编号</td><td>低浓度珠（@ 2.5×10 6珠/毫升） </td></tr><tr><td> EZ-Link的磺酸基-NHS的 - LC-生物素</td><td>赛默飞世尔</td><td> PI-21335 </td><td>未修改检测抗体生物素试剂盒</td></tr><tr><td> Tecan公司无限F200的读者</td><td> Tecan公司</td><td></td><td>酶标板读卡器</td></tr><tr><td>磷酸盐缓冲液</td><td> Sigma-Aldrich公司</td><td>的P-3688 </td><td> 1％PBS-BSA的，缓冲液; </td></tr><tr><td>一品脱的紧凑型超声波清洗机，115 VAC </td><td>科尔 - 帕默</td><td>吴-08849-00 </td><td>产生有效工作频率为55千赫</td></tr><tr><td>磁管分离器</td><td> Luminex公司</td><td> CN-0288-01 </td><td>对于单一1.5ml离心管中磁分离耦合的洗涤步骤</td></tr><tr><td>磁板分离器</td><td> Luminex公司</td><td> CN-0269-01 </td><td>对于96孔板磁分离检测洗涤步骤</td></tr></tbody></table>

conversion of a capture elisa to a luminex xmap assay using a multiplex antibody screening method

酶联免疫吸附试验（ELISA）长期以来的利益为生命科学研究和临床诊断的生物样品的分析物检测的主要工具。然而，酶联免疫吸附试验有其局限性。它通常在96孔板，井都涂有捕获抗体，需要的样本量相对较大，捕获抗原的利益。表面积大井和捕获抗体的疏水约束力，也可以导致非特异性结合和增加的背景。此外，大多数的ELISA依靠酶介导的信号放大，以达到合理的灵敏度。这种放大是不总是线性的和可倾斜的结果。 在过去的15年中，出现了一种新的技术，提供了酶联免疫吸附试验的好处，但也实现更高的吞吐量，增加灵活性，减少样本量，降低成本，具有类似禾rkflow 1，2。 Luminex液相xMAP技术是1（珠）微阵列平台，使双方monoplex和复用，可应用于蛋白质和核酸的应用3-5检测。珠捕获抗体共价固定在一个较小的表面积，需要较少的捕获抗体和样本量较小，比较酶联免疫吸附试验，非特异性结合是显著减少。与限制样品，如脑脊液，滑液等6个工作时，较小的样本量是重要的。复检测，进一步降低样本量的要求，使多个结果，从一个单一的样本。 最近由点阵仪的改进包括:新MAGPIX系统，体积更小，更便宜，更易于使用分析仪;低浓度MagPlex磁性微球的消除需要昂贵的过滤板，来更好的检测方法开发适合的工作浓度和低吞吐量应用; XMAP抗体偶联（ABC）的工具包，其中包括一项协议，试剂，消耗品耦合珠捕获抗体的利益所必需的。 （见材料第一个包内容的详细清单。） 在这个实验中，我们的XMAP平台转换为TNF-α的细胞因子的预优化的酶联免疫吸附试验和比较两种方法的性能7-11。 TNF-α是一种生物标志物用于测量与自身免疫性疾病患者的炎症反应。 我们开始由四个不同的微套或地区耦合四个候选人捕获抗体。当混合在一起，这四套允许有四个单独的抗体检测方法的所有四名候选人，以确定最佳的抗体对，节省试剂，样品和时间同步测试。 ，两个XMAP检测构建两个最优化的抗体对他们的表现相比原ELISA法检测到的信号强度，动态范围和灵敏度方面。

Watch this Scientific Journal Video about Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method at JoVE.com

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

ELISA方法可以很容易地转换到一个点阵仪XMAP法，通过复用，几种抗体的好处，可以同时进行筛选，以确定最佳的抗体对，导致更高的灵敏度和动态范围，同时降低检测成本。

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

conversion-capture-elisa-to-luminex-xmap-assay-using-multiplex

Research

JoVE Journal

Biology

43.5K 次观看.  Luminex Corporation. ELISA方法可以很容易地转换到一个点阵仪XMAP法，通过复用，几种抗体的好处，可以同时进行筛选，以确定最佳的抗体对，导致更高的灵敏度和动态范围，同时降低检测成本。

视频: Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

科研

免疫与感染

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

生物学

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

生物工程

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

血清中 疏螺旋体特异性 IgG 和 IgM 的多重免疫检测

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

作者聚焦:扩大用于莱姆疏螺旋体病诊断和病原体研究的多重免疫检测的范围 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

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基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

同时筛查1型糖尿病和多种自身免疫性疾病的高通量电化学发光7重检测